Modified Trichothecenes

2-Deoxy- 11-epi- 12-acetyl-3~t-hydroxysambucoin

2-Deoxy- 11-epi-3ct-hydroxysambucoin
Trichodiol A
Trichodiene
FS-1
FS-2
FS-3
FS-4
3-Ketoapotrichothecene
3ct-Hydroxyapotrichothecene
313-Hydroxyapotrichothecene

325
15. ModifiedTrichothecenes 327

Common/Systematic Name
2-Deoxy- 11-epi- 12-acetyl-3 a-hydroxysambucoin

Molecular Formula/Molecular Weight

C17H2604; M W = 294.18311

Me _H

OAc

M~Mel4~//2
14
OH
General Characteristics
Isolated as an oil.

Fungal Source
Fusarium sporotrichioides isolated from Ethiopian wheat.

Isolation/Purification
F. sporotrichioides was cultured on ground corn grits and the fermented corn grits were
blended with chloroform-acetone (85:15, v/v) and the mixture allowed to stand overnight.
After filtration the remaining solid residue was blended with acetone; the mixture was
filtered and the acetone extract and the chloroform-acetone extract were combined and
concentrated under reduced pressure, which yielded a dark green oil. The oil was
dissolved in 2 liters benzene-n-hexane (2:1, v/v) and aliquots were applied to Florisil
columns. Columns were eluted successively with benzene-n-hexane (2:1, v/v), methylene
chloride, chloroform-acetone (9:1, v/v), and acetone. The residue from the chloroform-
acetone fraction was recrystaUized from acetone-n-hexane to yield a mixture of
trichothecenes. Reversed-phase flash column chromatography of the solid trichothecene
mixture using MeOH-H20-acetic acid (35:20:1, v/v/v) yielded neosolaniol, NT-1, 8-
acetylneosolaniol, 8-propionyl-neosolaniol, 8-isobutyrylneosolaniol, 8-n-
butyrylneosolaniol, T-2 toxin, 8-n-pentanoyl-neosolaniol, and 8-n-hexanoylneosolaniol.
The acetone fraction was dissolved in benzene-acetone (2:1, v/v) and subjected to Florisil
column chromatography. The column was eluted successively with benzene-acetone (2:1,
v/v), chloroform-acetone (4:1, v/v), and acetone. The residue from the acetone fraction
was chromatographed using flash column chromatography, eluting with solvent mixtures
of increasing polarity: benzene-acetone (65:35, v/v), benzene-acetone (57:63, v/v),
acetone, acetone-methanol (1:1, v/v), and MeOH. Fractions were collected and similar
fractions were combined. The polar fractions of the silica gel column chromatography
were combined, concentrated at reduced pressure, and subjected to reversed-phase
column chromatography eluting with methanol-water-acetic acid (35:20:1, v/v/v). Similar
fractions were combined. Acuminatin, FS-1, scirpenol, T-2 tetraol, T-2 triol, 15-
acetylscirpenol, DON (deoxynivalenol), 8a-hydroxytrichothecolone, verrucarol, 2-deoxy-
11-epi-3 a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3 a-hydroxysambucoin were
328 15. ModifiedTrichothecenes

isolated in pure form.

Biological Activity
2-Deoxy- 11-epi-3a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3 ~-hydroxy-
sambucoin were screened for relative cytotoxicity in cultured baby hamster kidney (BHK'
21) cells and found to be non-toxic (LC~00=1000 ng/ml).

Spectral Data

IR:
(NaCI) 1736(acetyl group) and 3408cm"1 (OH group).

1H NMR:
(CDC13) 1.40(1H, dd, J=14.9, 2.0Hz, H-2); 2.32(1H, dd, J=14.9, 8.4Hz, H-2);
4.4(1H, m, H-3); 1.91(1H, m, H-4); 2.12(1H, m, H-4); 1.68(1H, dd, J=5.5, 2.0Hz, H-
7); 1.95(1H, dd, J=9.0, 2.0Hz, H-7); 1.95-2.0(2H, m, 2H-8); 5.45(1H, dd, J=6.0,
1.0Hz, H-10); 3.6(1H, bs, H-11); 3.58(1H, d, d=15.1Hz, H-13); 3.75(1H, d, d=15.1Hz,
H-13); 0.96(3H, s, H-14); 0.73(3H, s, H-15), 1.68(3H, s, H-16); and 2.05ppm (3H, s,
COCH3).

13C NMR:
(CDC13) 45.9, t, C-2; 67.5, d, C-3; 44.8, t, C-4; obscured by solvent, C-5; 35.2, s, C-
6; 24.5, t, C-7; 29.2, t, C-8; 140.4, s, C-9; 120.8, d, C-10; 75.9, d, C-11; 86.9, s, C-12;
74.5, t, C-13; 16.8, q, C-14; 16.3, q, C-15; 23.0, q, C-16; and 22.4ppm, q, COCH3.
(COCH3, not observed).

Mass Spectrum:
CIMS (TFA derivative): 391(M + + 1, 10%), 331(17), 301(38), 277(22), 217(55), and
111role (100).

Reference
D. M. Fort, C. L. Barnes, M. S. Tempestra, H. H. Casper, E. Bekele, A. A. Rottinghaus,
and G. E.Rottinghaus; Two New Modified Trichothecenes from Fusarium
sporotrichioides; J. Natural Products, Vol. 56, pp. 1890-1897(1993).
15. ModifiedTrichothecenes 329

Common/Systematic Name
2-Deoxy- 11-epi-3 a-hydroxysambucoin

Molecular Formula/M01ecular Weight

C15H2403; MW -- 2 5 2 . 1 7 2 5 4

Me H
16 9 .= O .

is M6 E..~.
14
OH
General Characteristics
Colorless crystals from methanol-H20, mp., 177-178 oC.

Funsal
v Source
Fusarium sporotrichioides isolated from Ethiopian wheat.

Isolation/Purification
F. sporotrichioides was cultured on ground corn grits at 10~ for 28 days and the
fermented corn grits were blended with chloroform-acetone (85:15, v/v) and the mixture
allowed to stand overnight. After filtration, the remaining solid residue was blended with
acetone. The mixture was filtered and the solid autoclaved and discarded. The acetone and
the chloroform-acetone extract were combined and concentrated under reduced pressure,
which yielded a dark green oil. The oil was dissolved in 2 liters benzene-n-hexane (2:1,
v/v), and aliquots were applied to Florisil columns. Columns were eluted successively with
benzene-n-hexane (2:1, v/v), methylene chloride, chloroform-acetone (9:1, v/v), and
acetone. The residue from the chloroform-acetone fraction was recrystallized from
acetone-n-hexane to yield a mixture of trichothecenes. Reversed-phase flash column
chromatography of the solid trichothecene mixture using methanol-water-acetic acid
(35:20:1, v/v/v) yielded neosolaniol, NT- 1, 8-acetylneosolaniol, 8-propionyl-neosolaniol,
8-isobutyrylneosolaniol, 8-n-butyrylneosolaniol, T-2 toxin, 8-n-pentanoylneosolaniol, and
8-n-hexanoylneosolaniol. The acetone fraction was dissolved in benzene-acetone (2:1, v/v)
and subjected to Florisil column chromatography. The column was eluted successively
with benzene-acetone (2:1, v/v), chloroform-acetone (4:1, v/v), and acetone. The residue
from the acetone fraction was chromatographed using flash column chromatography,
eluting with solvent mixtures of increasing polarity: benzene-acetone (65:35, v/v),
benzene-acetone (57:63, v/v), acetone, acetone-methanol (1:1, v/v), and MeOH. Fractions
were collected and similar fractions were combined. The polar fractions of the silica gel
column chromatography were combined, concentrated at reduced pressure, and subjected
to reversed-phase column chromatography eluting with methanol-water-acetic acid
(35:20:1, v/v/v). Similar fractions were combined. Acuminatin, FS-1, scirpenol, T - 2
tetraol, T-2 triol, 15-acetylscirpenol, DON (deoxynivalenol), 8a-hydroxytrichothecolone,
330 15. ModifiedTrichothecenes

verrucarol, 2-deoxy- 11-epi-3a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3tt-

hydroxysambucoin were isolated in pure form.

Biological Activity
2-Deoxy-11-epi-3a-hydroxysambucoin and 2-deoxy-11-epi-12-acetyl-3a-hydroxy-
sambucoin were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-
21) cells and found to be non-toxic (LC100= 1000ng/ml).

Spectral Data

IR:
(NaC1) 3412cm1 (OH group).

1H ~ :
(CDC13) 1.46(1H, dd, J-15.0, 2.4Hz, H-2); 2.28(1H, dd, .]=15.0, 9.4Hz, H-2);
4.44(1H, ddd, J=15.6,7.8, 2.4Hz, H-3); 1.85(1H, m, H-4); 2.02(1H, m, H-4); 1.53
(1H, dd, J-13.3, 2.0Hz, H-7); 1.91(1H, dd, J=8.0, 2.0Hz, H-7); 1.95-2.0(2H, m, H-
8); 5.52(1H, dd, J=5.6, 0.8Hz, H-10); 3.69(1H, bs, H-11); 3.66(1H, d, J=12.6Hz, H-
13); 3.95(1H, d, J=12.6Hz, H-13); 0.97(3H, s, 3H-14); 0.71(3H, s, H-15); and
1.70ppm (3H, s, H-16).

13C NMR:
(CDC13) 44.1, t, C-2; 67.0, d, C-3; 44.2, t, C-4; 48.6, s, C-5; 35.0, s, C-6; 24.3, t, C-7;
28.2, t, C-8; 139.5, s, C-9; 119.5, d, C-10; 75.1, d, C-11; 77.6, s, C-12; 74.0, t, C-13;
16.6, q, C-14; 16.1, q, C-15; and 23.0ppm, q, C-16.

Mass Spectrum:
LREIMS (TMSi derivative): 396(NY, 1%), 378(4), 258(100), 108(63), and 73m/e
(60); ElMS (TMSi derivative): 397(M++ 1, 18%), 381 (15), 307(22), 291 (100),
263(42), 217(53), 199(32), 73role (21); and CIMS (TFA derivative): 445(M § + 1,
45%), 445(45), 331(100), 217(13), 115(67), and 93role (80).

Reference
D. M. Fort, C. L. Barnes, M. S. Tempestra, H. H. Casper, E. Bekele, A. A. Rottinghaus,
and G. E. Rottinghaus; Two New Modified Trichothecenes from Fusarium
sporotrichioides; J. Natural Products, Vol. 56, pp. 1890-1897(1993).
15. ModifiedTrichothecenes 331

Common/Systematic Name
Trichodiol A

Molecular Formula/Molecular Weight

C15H2403,1VIW' = 252.17254

Me Me~/.O H

General Characteristics
Colorless crystals from ether-hexane; mp., 81-830C; [tg]D = +52 ~ (CHC13).

Isolation/Purification
The ethyl acetate extract of the fermentation broth from Trichothecium r o s e u m was
saponified with 10% ethanolic KOH at room temperature. The mixture was concentrated
to ca. 1/3 volume below 40~ under reduced pressure. This material was extracted with
ether and the extract washed with water, dried and the solvent evaporated to dryness.
Repeated column chromatography of the nonsaponifiable material using Wakogel C-200
(60% ether-benzene) gave trichodiol A which crystallized from ether-hexane to give a
pure sample.

Fungal Source
T r i c h o t h e c i u m roseum.

Spectral Data

UV:
E~H End absorption, 210nm (e = 360).

IR:
(CHCI3) 3460cm1 (OH).

1H NMR:
(CDC13) The NMR spectrum revealed the presence of three tertiary methyl
groups (singlets at 0.86, 0.96, and 1.15ppm, 3H each), a CHzOH group (3.48,
4.06, 2H, Abq, J = 12.0Hz) attached to a non-hydrogen atom, and a-CH=CH-
group (5.46, 5.73ppm, 2H, Abq, J = 10Hz). Both carbon atoms are attached to
quaternary carbon atoms and a methine proton (3.17ppm, 1H, bs). The signals at
3.48 and 4.06ppm shifted to 4.33 and 4.54ppm upon acetylation.

Mass Data:
HREIMS: 234.162(C15H2202, M § - 1-120, calcd. 234.162), 127.074(C7Hl102, calcd
127.076), 125.099(CsHx30, ealcd 125.097), 108.095(CsH12, calcd 108.094),
107.088(C8H11, calcd 107.086), 109.065(C7H90, calcd 109.065), 9 6 . 0 5 6 m / e (C6HsO,
calcd 96.058). Found: O, 18.84; C15H2403 requires O, 19.02%.
332 15. ModifiedTrichothecenes

Reference
S. Nozoe and Y. Machida; The Structures of Trichodiol and Trichodiene; Tetrahedrort,
Vol. 28, pp. 5105-5111(1972).
15. ModifiedTrichothecenes 333

Common/Systematic Name
Trichodiene

Molecular Formula/Molecular Weight

C15H24; M W --" 204.18780

CH2

General Characteristics
Colorless oily material; [a]D--" q- 21 ~ (CHCI3).

Isolation/Purification
The myeelium from the fermentation of T. r o s e u m was suspended in warm acetone for 3
hours. After filtration, the mycelium was resuspended in acetone and allowed to stand
overnight at room temperature and then filtered. The combined acetone extracts were
concentrated to ca. ~/3 volume, to which water was added. Extraction with ethyl acetate
followed by successive washing with water, drying, and removal of solvent afforded a
brown residue, which was saponified with 10% KOH at room temperature to obtain the
nonsaponifiable material. Column chromatographic separation was first afforded using
ether-benzene. The first fraction (100% benzene) was further chromatographed using a
AgNO3 impregnated silica gel column (hexane elution) to obtain pure trichodiene.

Fungal Source
Trichothecium roseum.

Spectral Data

IR:
(CHC13) 3065, 1645, and 890cm"].

]H NMR:
(CDCI3) The NMR spectrum revealed the presence of ringlets at 0.85 and 1.04 ppm
due to two tertiary methyl groups, a broad ringlet at 1.63ppm due to an olefinic methyl
group, a multiplet at 5.23ppm due to an olefinic proton, and two broad singlets at 4.71
and 4.92ppm due to olefinic protons.

Mass Spectrum:
EIMS: 204(M +, C15H24); intense peaks at 189, 161,133, 121, 119, 109 (base peak
CsHI3), 95 (Cyril0, 93, and 67m/e.
334 15. Mo d i fi ed T r i ch o th ecen es

Reference
S. Nozoe and Y. Machida; The Structures of Trichodiol and Trichodiene; Tetrahedron,
Vol. 28, pp. 5105-5111(1972).
15. ModifiedTrichothecenes 335

Common/Systematic Name
FS-1

Molecular Formula/Molecular Weight

C15H2203; MW = 250.15689

16 ,,OH
~,' 13CH20H

0
General Characteristics
Isolated as an oil.

Fungal Source
Fusarium sporotrichioides (MC-72083) and F. sambucinum.

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil
column chromatography eluted with benzene-hexane, 2:1 (v/v), methylene chloride, and
chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil
was further purified by normal phase preparative HPLC using benzene-acetone followed
by preparative reversed-phase TLC to yield several trichothecenes including FS-1.

Spectral Data

IR~
(thin film) 1684(enone) and 3352cm"1 (OH).

1H NMR:
(CDC13) 6.24(1H, t, J-1.5Hz, H-2); 3.10(1H, d, J-18.2Hz, H-4), 2.03(1H, d,
J=18.2Hz, H-4); 1.95(1H, dd, ,/=15.0, 4.6Hz, H-7); 1.79(1H, d, J=15.0Hz, H-7);
1.45(2H, m, J-4.6Hz, H-8); 5.05(1H, br s, H-10); 4.11(1H, br s, H-11); 4.55(1H, d,
J= 17.3I-Iz,
H-13); 4.34(1H, d, J-17.3Hz, H-13); 1.24(3H, s, CH3-14); 0.84(3H, s, CH3-15); and
1.61ppm (3H, br s, CH3-16).

13C NMR:
(CDC13) 129.5, C-2; 208.4, C-3; 50.4, C-4; 53.1, C-5; 41.9, C-6; 29.9, C-7; 27.4,
C-8; 135.7, C-9; 125.0, C-10; 71.3, C-11; 188.0, C-12; 61.0, C-13; 22.4, C-14; 13.1,
C-15; and 22.5ppm; C-16.
336 15. ModifiedTrichothecenes

Mass Spectrum:
HREIMS: 250.150m/e (M+); calcd for C15H2zO3,250.156; LREIMS: 250, 232, 219,
204, 167(base peak), 126, 107, 84, and 55m/e.

References
D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene
Mycotoxins of Fusarium sporotrichioides (MC- 72083); Tet. Lett., pp. 4133-4136
(1986).

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of

Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other
Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257
(1994).
15. ModifiedTrichothecenes 337

Common/Systematic Name
FS-2

Molecular Formula/Molecular Weight

C15H2403, M W = 2 5 2 . 1 7 2 5 4

16
9 All
HO,,,"~ "~ 13CH20H

OH
Fungal Source
Fusarium sporotrichioides (MC-72083).

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by
acetone. The combined extracts were concentrated to yield a dark red oil which was
treated with n-hexane to remove non-polar materials. The resulting orange-red oil was
further purified by Florisil column chromatography eluted with benzene-hexane, 2:1 (v/v),
methylene chloride, and chloroform-methanol (95:5, v/v) followed by acetone. The latter
two were combined to give an oil highly enriched with trichothecenes. Flash
chromatography followed by normal phase preparative HPLC using toluene-acetone and
preparative reversed-phase TLC yielded several purified triehotheeenes including FS-2.

Spectral Data

UV:
max 194.5nm (e=12,000; two ene 7: to re* transitions).

:H NMR:
(CDCI3) 5.75(1H, dd, J=l.5, 3.3Hz, H-2); 4.79(1H, m, H-3), 2.10(1H, m, H-4);
1.75(1H, m, H-7); 1.31(1H, m, H-7); 1.98, 1.73(2H, m, H-8); 5.58(1H, d, J=10.EHz,
n-10); 5.67(1H, dd, J=l.7, 10.2Hz, n-11); 4.35(1H, br d, J=14.5Hz, n-13); 4.23(1H,
br d, J=14.5Hz, H-13); 1.09(3H, s, CH3-14); 0.95(3H, s, CH3-15); and 1.27ppm (31-1,
s, CH3-16).

13C N M R :
(CDCI3) 133.0, C-2, 73.4, C-3; 47.4, C-4, 54.6, C-5, 40.0, C-6; 27.8, C-7, 35.0, C-8;
65.7, C-9, 135.6, C-10, 133.2, C-11,154.0, C-12; 60.7, C-13; 21.3, C-14, 22.0, C-15;
and 30.9ppm (C- 16).
 338 15. ModifiedTrichothecenes

Mass Spectrum:
HREIMS: 252.173m/e (M+); calcd for C15H2403,252.173. CIMS: 253(M § + 1, 2%),
235(M + - OH, 50), 127(cleavage ofC-6/C-6 bond, 85), 125(90), 109(127 - I410, 100),
and 107m/e (125 - 1-/20, 50),

References
D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene
Mycotoxins ofFusarium sporotrichioides (MC- 72083); Tet. Lett., pp. 4133-4136
(1986).

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of

Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other
Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257
(1994).
15. Modified Trichothecenes 339

Common/Systematic Name.
FS-3

Molecular Formula/Molecular Weight

C 1 5 H 2 0 0 3 ; M W -- 248.14124

16 0
13CH20H

General Characteristics
Isolated as a colorless glass.

Fungal Source
Fusarium sambucmum.

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over-
night, filtered and the solid residue re-extracted with acetone. The acetone extract was
combined with the chloroform-acetone extract, concentrated under vacuum, and the non-
polar material~were removed by hexane drip. The solvent was decanted, concentrated and
chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1"1
(v/v), acetone, and acetone-methanol, 11 (v/v). The toluene-acetone (4:1, v/v) fraction
was further purified by flash chromatography eluting with toluene-acetone (4:1v/v). This
resulted in purified diacetoxyscirpenol, 3,15-diacetoxyscirpenol, 3,4,15-
triacetoxyscirpenol, neosolaniol, sambucoin, 4-monoacetoxyscirpenol, 15-
monoacetoxyscirpenol, and FS-3.

Spectral Data

UV:
max^~176 229nm (e = 13,000).

IR:
(film) 3470(OH)and 1675crn1 (C=O).

'H NMR:
(CDCI3) 6.29(IH, t,J=1.5I-Iz,H-2); 2.32(11-1,d, J=18.8, H-4), 2.60(IH, d,
J=I8.8Hz, H-4); 1.73(IH, dd, J=I2.0, 2.5Hz, H-7); 1.87(IH, dd, J=I2.0, 2.51-Iz,H-7);
2.20(IH, dd, J=19.0, 2.51-1z,H-8); 1.63(IH, dd, J=19.0, 2.SHz, H-8); 5.75(IH, br s,
H-10), 4.49(IH, dd, d=I7.0, 1.5Hz, H-13), 4.56(IH, dd, J=17.0, 1.5Hz, H-13),
1.50(3H, s, CH3-14); 1.17(3H, s, CH3-15); and 1.93ppm (3H, s, CH3-16).
 340 15. ModifiedTrichothecenes

13C NMR:
(CDC13) 129.6, C-2; 207.0, C-3; 50.6, C-4; 53.3, C-5; 47.1, C-6; 30.9, C-7; 28.3, C-
8; 160.1, C-9; 126.4, C-10; 202.5, C-11; 186.3, C-12; 61.6, C-13; 23.8, C-14; 18.2, C-
15; and 29.0ppm; C- 16.

Mass Spectrum:
HREIMS: 248.141m/e (M+); calcd for C15H2003,248.154; LREIMS: 248(M+, 1%),
181(14), 131(31), 124(26), and 69m/e (100).

References
D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta,
and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem.,
Vol. 54, pp. 4313-4318(1989).

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of

Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other
Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257
(1994).
15. Modified Trichothecenes 341

Common/Systematic Name
FS-4

Molecular Formula/Molecular Weight

C15H2203; M W -- 2 5 0 . 1 5 6 8 9

16

HO"~ 13CH2H
0

0
General Characteristics
Isolated as a colorless glass.

Fungal Source
Fusarium sambucinum.

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over-
night, filtered and the solid residue re-extracted with acetone. The acetone extract was
combined with the chloroform-acetone extract, concentrated under vacuum and the non-
polar materials removed by hexane drip. The solvent was decanted, concentrated and
chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1
(v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction
was further purified by preparative reversed-phased HPLC with water-acetonitrile (5:1,
v/v) as mobile phase. This resulted in purified FS-4.

Spectral Data

UV:
,~ maxACa~ 198nm (e = 10,400 rr - ~:* transition).

IR:
(film) 3450(OH) and 1690cm1 (C--O).

1H N~IR:
(CDCla) 6.27(1H, t, J=l.5Hz, H-2); 2.15(1H, d, ,/--19.0, H-4), 2.73(1H, d, J=19.0Hz,
H-4); 1.40(1H, m, n-7); 1.70(1H, m, n-7); 1.70(1H, m, n-8); 1.85(1H, m, n-8);
5.25(1H, d, J=10.0Hz, H-10); 5.61(1H, dd, J=10.0, 1.5Hz, H-11); 4.69(1H, d, J--17.0,
n-13); 4.35(1H, d, J=17.0, n-13); 1.29(3H, s, CH3-14); 0.98(3H, s, CHa-15); and
1.25ppm (3H, s, CH3-16).
342 15. ModifiedTrichothecenes

13C NMR:
(CDC13) 128.1, C-2; 205.0, C-3; 49.0, C-4; 28.2, C-7; 32.5, C-8; 133.6, C-10; 130.9,
C-11; 183.0, C-12; 62.0, C-13; 19.2, C-14; 18.0, C-15; and 29.0ppm (C-16).

Mass Spectrum:
EIMS: 235(4%, M + - Me), 125(86), 108(100), and 81m/e (83); exact mass for
C8H130 ( M + - C7H902) calcd. 125.097; found 125.096m/e; C7H902 ( M + - C s n l 3 0 )
calcd 125.060, found 125.059; CsH11 (CsH130 - 1-120)calcd. 107.086, found 107.086.

References
D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta,
and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem.,
Vol. 54, pp. 4313-4318(1989).

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of

Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other
Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257
(1994).
15. ModifiedTrichothecenes 343

Common/Systematic Name
3-Ketoapotrichothecene

Molecular Formula/Molecular Weight

C15H2203; M W -" 2 5 0 . 1 5 6 8 9

13

H CH2OH

15 14

General Characteristics
Colorless glass.

Fungal Source
Fusarium sambucmum.

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over
night, filtered and the solid residue re-extracted with acetone. The acetone extract was
combined with the chloroform-acetone extract, concentrated under vacuum and the non-
polar materials removed by hexane drip. The solvent was decanted, concentrated and
chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1
(v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (2:1, v/v) fraction
was further purified by preparative normal-phase TLC which resulted in purified 3-
ketoapotrichothecene.

Spectral Data

UV:
~. maxA'~~176 197nm(e=8,600 rr to n* transition).

Im:
(film) 3420(OH) and 1737cm1 (C=O).

1H NMR:
(CDC13) 2.60(1H, dd, J=20.0, 2.2Hz, H-2); 2.79(1H, dd, J=20.0, 1.4Hz, H-2);
2.14(1H, dd, J=19.2, 1.4Hz, H-4), 2.68(1H, dd, J=19.2, 2.2Hz, H-4); 1.48(1H, m, H-
7); 1.63(1H, m, H-7); 2.05, 2.15(2H, m, H-8); 5.55(1H, m, H-10); 4.23(1H, m, H-11);
3.53(1H, d, J=l 1.3Hz, H-13); 3.83(1H, d, J=l 1.3Hz, H-13); 1.16(3H, s, CH3-14);
0.76(3H, s, CH3-15); and 1.68ppm (3H, s, CH3-16).
344 15. ModifiedTrichothecenes

13CNMR:
(CDC13) 49.6, C-2; 215.5, C-3; 49.6, C-4; 51.3, C-5; 46.2, C-6; 28.8, C-7; 26.8, C-8;
135.7, C-9; 210.4, C-10; 80.1, C-11; 90.8, C-12; 65.0,.C-13; 19.2, C-14, 15.3, C-15;
and 22.6ppm (C- 16).

Mass Spectrum:
HR IMS: 250.158m/e (M+); calcd for C15H2203,250.156. LREIMS: 250(M+,15%),
235(38), 124(45), 107(50), and 43m/e (100).

References
D. R. Sanson, D. G. Codey, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta,
and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem.,
Vol. 54, pp. 4313-4318(1989).

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of

Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other
Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257
(1994).
15. ModifiedTrichothecenes 345

Commort/Systematic Name
3 a-Hydroxyapotrichothecene
3a, 13-Dihydroxy- 11-epiapotrichothec-9-ene

Molecular Formula/Molecular Weight

C15H2403; MW = 252.17254

13
H CH2OH

15 14

General Characteristics
Crystals from 2-propanol-hexane; mp., 167-169~ [a]D - 33.2 ~ (in ~EtOH).

Fungal Source
Fusarium sambucmum, F. graminearum (ATCC 28114), F. crookwellense, F. culmorum,
and F. sporotrichioides.

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over
night, filtered and the solid residue re-extracted with acetone. The acetone extract was
combined with the chloroform-acetone extract, concentrated under vacuum and the non-
polar materials removed by hexane drip. The solvent was decanted, concentrated and
chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1
(v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction
was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which
resulted in purified 3a-hydroxyapotrichothecene. Alternatively, the crude extract from F.
graminearum was chromatographed on Florisil; after removal of 3-aeetyldeoxynivalenol,
an oil remained which was chromatographed by medium-pressure liquid chromatography
using LiChroprep silica gel column eluted with the following: 10% ethyl acetate-hexane;
20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. The later fractions
eluting with ethyl acetate contained the 3a and 3 [3-isomers; these were separated by
preparative HPLC using a CSC-S nitrile column with a 6% 2-propanol-hexane mobile
phase and a flow rate of 4 ml/minute.

Spectral Data

UV~
~ Aoetonilrilr
max 196nm (e=7,200 ~ to rr* transition).
 346 15. ModifiedTrichothecenes

IR:
(film) 3400cm1 (OH).

1H NMR:
(CDC13) 1.74(1H, m, H-2); 2.62(1H, ddd, J-1.7, 6.1, 12.3Hz, H-2); 4.30(1H, b m,
H-3), 1.64(1H, m, H-4); 2.15(1H, dd, J=10.3, 13.0Hz, H-4); 1.40(1H, dddd, J=2.0,
4.0, 5.8, 13.0Hz, H-7); 1.58(1H, m, H-7); 2.02(1H, m, H-8); 1.62(1H, b m, H-8);
5.55(1H, b sptet, H-10); 4.18(1H, b sptet, H-11); 3.18(1H, b t, J=l 1.1Hz, H-13);
3.77(1H, dd, J=8.0, ll.lHz, H-13); 1.02(3H, s, CH3-14); 0.95(3H, s, CH3-15); and
1.50ppm (3H, q,J=l.4Hz, CH3-16).

13C NMR:
(CDC13) 44.4, C-2; 72.8, C-3; 43.5, C-4; 52.5, C-5; 44.7, C-6; 27.8, C-7; 29.3, C-8;
135.5, C-9; 21.4, C-10; 81.3, C-11; 92.5, C-12; 63.3, C-13; 19.2, C-14; 17.9, C-15;
and 22.6ppm (C-16).

Mass Spectrum:
HREIMS: 252.173m/e (M+); calcd for C15H2403, 252.186. LREIMS: 252(M § ,
28%), 237(17), 140(100), and 124role (51).

References
R. Greenhalgh, D. A. Fielder, L. A. Morrison, J-P Charland, B. A. Blaekwell, M. E.
Savard, and J. W. Apsimon; Secondary Metabolites of Fusarium species:
Apotriehothecene Derivatives; J. Agile. food Chem., Vol. 37, pp. 699-705(1989).

D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta,

and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem.,
Vol. 54, pp. 4313-4318(1989).

M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of

Secondary Metabolites from Fusarium Fungi, In Mycotoxins in Grain; Compounds Other
Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257
(1994).
15. ModifiedTrichothecenes 347

Common/Systematic Nam_e
313-Hydroxyapotrichothecene

Molecular Formula/Molecular Weight

C15H2403; M W = 252.17254

13
H ,H2OH

15 14

General Characteristics
Colorless glass.

Fungal Source
Fusarium sambucinum.

Isolation/Purification
Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over
night, filtered and the solid residue re-extracted with acetone. The acetone extract was
combined with the chloroform-acetone extract, concentrated under vacuum and the non-
polar materials removed by hexane drip. The solvent was decanted, concentrated and
chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1
(v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction
was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which
resulted in purified 3[3-hydroxyapotrichothecene.

Spectral Data

UV:
~. maxA~176176196nm(e=7,800 z~to n* transition).

IR:
(film) 3420 and 1048cm1 (-C-O-C-).

1H NMR:
(CDC13) 1.38(1H, m, H-2); 2.45(1H, ddd, 3"=1.3, 5.8, 6.4Hz, H-2); 4.51(1H, b m, H-
3), 2.10(1H, H-4); 2.23(1H, H-4); 1.36(1H, m, H-7); 1.57(1H, m, H-7); 1.99(2H, m,
H-8); 5.50(1H, H-10); 4.11(1H, H-11); 3.56(1H, d, J=l 1.4Hz, H-13); 3.78(1H, d,
J=l 1.4Hz, H-13); 1.07(3H, s, CH3-14); 0.52(3H, s, CH3-15); and 1.63ppm (3H, b s,
CH3-16).
348 15. ModifiedTrichothecenes

13C NMR:
(CDC13) 47.0, C-2; 74.2, C-3; 45.7, C-4; not observed, C-5; not observed, C-6; 27.5,
C-7; 29.3, C-8; not observed, C-9; 122.5, C-10, 81.3, C-11; not observed, C-12; 65.5,
C-13; 20.0, C-14; 16.5, C-15; and 22.5ppm (C-16).

Mass Spectrum:
HREIMS: 252.181m/e (M+); calcd for C15H2403,252.186. LREIMS: 252(M~, 15%),
237(15), 124(38), 107(92), and 83m/e (100).

Reference
D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta,
and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem.,
Vol. 54, pp. 4313-4318(1989).